Chromenopyrazoles: Non-psychoactive and Selective CB1 Cannabinoid Agonists with Peripheral Antinociceptive Properties

Article abstract of DOI:10.1002/cmdc.201100568

The unwanted psychoactive effects of cannabinoid receptor agonists have limited their development as medicines. These CB₁-mediated side effects are due to the fact that CB₁ receptors are largely expressed in the central nervous system (CNS). As it is known that CB₁ receptors are also located peripherally, there is growing interest in targeting cannabinoid receptors located outside the brain. A library of chromenopyrazoles designed analogously to the classical cannabinoid cannabinol were synthesized, characterized, and tested for cannabinoid activity. Radioligand binding assays were used to determine their affinities at CB₁ and CB₂ receptors. Structural features required for CB₁/CB₂ affinity and selectivity were explored by molecular modeling. Some compounds in the chromenopyrazole series were observed to be selective CB₁ ligands. These modeling studies suggest that full CB₁ selectivity over CB₂ can be explained by the presence of a pyrazole ring in the structure. The functional activities of selected chromenopyrazoles were evaluated in isolated tissues. Invivo behavioral tests were then carried out on the most effective CB₁ cannabinoid agonist, 13a. Chromenopyrazole 13a did not induce modifications in any of the tested parameters on the mouse cannabinoid tetrad, thus discounting CNS-mediated effects. This lack of agonistic activity in the CNS suggests that this compound does not readily cross the blood-brain barrier. Moreover, 13a can induce antinociception in a rat peripheral model of orofacial pain. Taking into account the negative results obtained with the hot-plate test, the antinociception induced by 13a in the orofacial test could be mediated through peripheral mechanisms.

Full text of DOI:10.1002/cmdc.201100568

MED
DOI: 10.1002/cmdc.201100568
Chromenopyrazoles: Non-psychoactive and Selective CB1
Cannabinoid Agonists with Peripheral Antinociceptive
Properties
Jose Cumella,[a] Laura Hernꢀndez-Folgado,[a] Rocio Girꢁn,[b] Eva Sꢀnchez,[b] Paula Morales,[a]
Dow P. Hurst,[c] Maria Gꢁmez-CaÇas,[d] Maria Gꢁmez-Ruiz,[d] Diana C. G. A. Pinto,[e]
Pilar Goya,[a] Patricia H. Reggio,[c] Marꢂa Isabel Martin,[b] Javier Fernꢀndez-Ruiz,[d]
Artur M. S. Silva,[e] and Nadine Jagerovic*[a]
The unwanted psychoactive effects of cannabinoid receptor
agonists have limited their development as medicines. These
CB1-mediated side effects are due to the fact that CB1 recep-
tors are largely expressed in the central nervous system (CNS).
As it is known that CB1 receptors are also located peripherally,
there is growing interest in targeting cannabinoid receptors lo-
cated outside the brain. A library of chromenopyrazoles de-
signed analogously to the classical cannabinoid cannabinol
were synthesized, characterized, and tested for cannabinoid ac-
tivity. Radioligand binding assays were used to determine their
affinities at CB1 and CB2 receptors. Structural features required
for CB1/CB2 affinity and selectivity were explored by molecular
modeling. Some compounds in the chromenopyrazole series
were observed to be selective CB1 ligands. These modeling
studies suggest that full CB1 selectivity over CB2 can be ex-
plained by the presence of a pyrazole ring in the structure.
The functional activities of selected chromenopyrazoles were
evaluated in isolated tissues. In vivo behavioral tests were then
carried out on the most effective CB1 cannabinoid agonist,
13a. Chromenopyrazole 13a did not induce modifications in
any of the tested parameters on the mouse cannabinoid
tetrad, thus discounting CNS-mediated effects. This lack of ag-
onistic activity in the CNS suggests that this compound does
not readily cross the blood–brain barrier. Moreover, 13a can
induce antinociception in a rat peripheral model of orofacial
pain. Taking into account the negative results obtained with
the hot-plate test, the antinociception induced by 13a in the
orofacial test could be mediated through peripheral mecha-
nisms.
Introduction
The three major components of marijuana are D9-tetrahydro-
cannabinol (D9-THC), cannabidiol (CBD), and cannabinol
(CBN).[1] Unlike D9-THC,[2] CBD and CBN are non-psychotropic
phytocannabinoids.[3] D9-THC interacts with two well-character-
ized G-protein-coupled receptors: CB1 and CB2.[4,5] The CB1 re-
ceptors are localized with high density in the brain and are
also found in peripheral tissues. In contrast, the CB2 receptors
are expressed mainly in immune cells, although they can also
be found in the CNS, particularly under pathological circum-
stances. The activity of the cannabinoid receptors is elicited
not only by phytocannabinoids, but also by synthetic ligands
and endocannabinoids.[6–9] The only cannabinoid receptor li-
gands prescribed so far are CB1/CB2 receptor agonists. Cesamet
(nabilone) and Marinol (D9-THC) are used for the treatment of
nausea and vomiting associated with cancer chemotherapy or
as anti-emetic agents. Sativex (D9-THC and CBD) is prescribed
to relieve spasticity and pain in patients of multiple sclerosis.
However, preclinical data indicate that CB1 and/or CB2 receptor
agonists are useful for diverse therapeutic applications, includ-
ing pain relief, treatment of intestinal disorders, glaucoma,
cancer proliferation, and neurodegenerative diseases.[10–12]
D9-THC and CBN are classical cannabinoids characterized by
tricyclic terpenoids bearing a benzopyran moiety (Figure 1).[13]
Structure–activity relationship (SAR) studies of D9-THC and D8-
THC analogues for CB1/CB2 receptors have been widely report-
[a] Dr. J. Cumella, Dr. L. Hernꢀndez-Folgado, P. Morales, Prof. Dr. P. Goya,
Dr. N. Jagerovic
Instituto de Quꢁmica Mꢂdica, CSIC
Juan de la Cierva 3, 28006 Madrid (Spain)
E-mail: nadine@iqm.csic.es
[b] Dr. R. Girꢃn, Dr. E. Sꢀnchez, Prof. Dr. M. I. Martin
Departamento de Farmacologꢁa y Nutriciꢃn
Facultad de Ciencias de la Salud, Universidad Rey Juan Carlos
Avda. Atenas S/N, 28922 Alcorcꢃn, Madrid (Spain)
[c] Dr. D. P. Hurst, Prof. Dr. P. H. Reggio
Department of Chemistry and Biochemistry
University of North Carolina Greensboro
Greensboro, NC 27402 (USA)
[d] M. Gꢃmez-CaÇas, Dr. M. Gꢃmez-Ruiz, Prof. Dr. J. Fernꢀndez-Ruiz
Departamento de Bioquꢁmica y Biologꢁa Molecular, Facultad de Medicina
Centro de Investigaciꢃn Biomꢂdica en Red sobre Enfermedades
Neurodegenerativas (CIBERNED)
Instituto Ramꢃn y Cajal de Investigaciꢃn Sanitaria (IRYCIS)
Universidad Complutense de Madrid, 28040 Madrid (Spain)
[e] Dr. D. C. G. A. Pinto, Prof. Dr. A. M. S. Silva
Department of Chemistry & QOPNA
University of Aveiro, 38210-193 Aveiro (Portugal)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100568.
452
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Chromenopyrazoles
ing resorcinol was allowed to react with 3,3-dimethylacrylic
acid in methanesulfonic acid in the presence of phosphorus
pentoxide to form the 7-alkyl-5-hydroxy-2,2-dimethylchroman-
4-ones 3 and 4 under microwave heating conditions and using
the reagents described by Lim et al.[31] For the a-formylation of
3 and 4, an alternative procedure to the overnight heating
proposed by Press and colleagues[32] yielded the corresponding
Figure 1. Structures of D9-THC, D8-THC and cannabinol derivatives.
ed.[14–19] It is well established that the C1, C3, and C9
positions play a key role in the binding affinity and
pharmacological potency of THCs. Although some of
these classical cannabinoids have been reported to
show significant selectivity for one of the two recep-
tor types, structural variations within D9-THC have
generally resulted in derivatives with high affinity for
both CB1 and CB2 receptors. Considerable effort has
been directed toward the SARs of THCs; however,
fewer structural modifications have been made on
the structure of cannabinol. Rhee et al.[20] reported
the binding and inhibition of adenylyl cyclase by a
series of CBN derivatives. Unlike CBN, which was
found to be less potent than D9-THC, the 3-dimethyl-
heptyl CBN analogue and 9-hydroxymethyl CBN ana-
logues showed higher affinity and agonist potency
than D9-THC at both CB1 and CB2 receptors. The pres-
ence of alkyl or aryl esters at position 9 of CBN result-
ed in weak CB1 and CB2 binding.[21] Cannabilactones
were reported more recently by Khanolkar et al.[22]
One of them exhibited high CB2 receptor affinity,
Scheme 1. Reagents and conditions: a) 3,3-dimethylacrylic acid, CH3SO3H, P2O5, 708C, MW,
10 min; b) NaH, THF, MW, 468C, 20 min, then ethyl formate, THF, MW, 468C, 20 min;
c) H2N-NHR2, EtOH, 16 h, RT or 10 min, MW.
with 500-fold selectivity over the CB1 receptor. All these consid-
erations taken together, it is clear that the structural require-
ments for cannabinoid receptor binding by the CBN series dif-
fers from those of the THCs.
(Z)-7-alkyl-5-hydroxy-3-(hydroxymethylen)-2,2-dimetylchroman-
4-ones 5 and 6 in 20 min by using microwave irradiation. Con-
densation of the b-keto aldehydes 5 and 6 with the appropri-
ate hydrazine then gave the corresponding 7-alkyl-1(2),4-dihy-
dro-4,4-dimethylchromeno[4,3-c]pyrazol-9-oles 7–15. From
methyl- and ethylhydrazines, the two N1- and N2-substituted
pyrazole isomers (8a,b; 9a,b; 13a,b) were isolated with an ap-
proximate relative ratio from 8:2 to 6:4 (N1/N2). However, reac-
tion of b-keto aldehyde 5 or 6 with arylhydrazine resulted in
only one isomer being isolated, corresponding to the N1-aryl-
chromenopyrazole (10a, 14a, and 15a). The fact that alkylhy-
drazines give a mixture of N1- and N2-substituted 7-alkyl-1(2),4-
dihydro-4,4-dimethylchromeno[4,3-c]pyrazol-9-oles can be ex-
plained by the reaction of N’-hydrazine as well as N-hydrazine
with the aldehyde group of 5 and 6, giving compounds 8a,b,
9a,b, and 13a,b upon cyclization. For N’-arylhydrazines, N’-hy-
drazine is much less nucleophilic than N-hydrazine, and this
leads to a single isomer, as in the case of 10a, 14a, and 15a.
The pharmacological properties of CBN have also received
less attention than the THCs. Analgesic properties of CBN have
been reported in various models of pain.[23–26] CBN generally re-
quires higher doses than D9-THC to produce antinociception,
but it shows minimal psychomimetic effects.[27,28] One of the
main challenges in the design of new cannabinoid ligands is
the avoidance of CNS side effects. In 1985 Press and Birn-
berg[29] reported benzopyrano[4,3-c]pyrazoles that did not
show neuroleptic activity in locomotor and catalepsy tests. We
propose exploration of this scaffold for cannabinoid ligands. In
this context, we report herein the contribution of a pyrazole
ring in place of the cannabinol phenyl group toward cannabi-
noid activity.
Results
Biological assays
Chemistry
Competitive binding studies
7-Alkyl-1,4-dihydro-4,4-dimethylchromenopyrazol-9-oles 7–15
were prepared from the corresponding resorcinol as shown in
Scheme 1. 5-(1’,1’-Dimethyl-n-heptyl)-1,3-dihydroxybenzene (2)
was previously synthesized by demethylation[30] of 5-(1’,1’-di-
methyl-n-heptyl)-1,3-dimethoxybenzene. The appropriate start-
The compounds reported herein were evaluated in vitro for
their ability to displace [3H]CP55940 from human cannabinoid
CB1 and CB2 receptors transfected into HEK293 EBNA cells.
They were first subjected to a preliminary screen at a concen-
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N. Jagerovic et al.
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Table 1. Binding affinity of chromenopyrazole derivatives 7–15 for hCB1 and hCB2 cannabinoid receptors.
[c]
Compd
R1
R2
hCB1 Ki [nm][a]
hCB2 Ki [nm][a]
CB1/CB2
7
8a
8b
9a
9b
10a
11
12b
13a
pentyl
pentyl
pentyl
pentyl
pentyl
H
4100ꢀ800
4700ꢀ1200
22100ꢀ1410
9610
2010ꢀ500
3460ꢀ1000
>40000
>40000
4450ꢀ1015
>40000
>40000
>40000
>40000
>40000
270
0.5
0.7
1-methyl
2-methyl
1-ethyl
2-ethyl
–
–
–
>40000
pentyl
1-(3,4-dichlorophenyl)
607ꢀ151
28.5ꢀ33.6
14.2ꢀ4.2
4.5ꢀ0.8
–
1,1-dimethylheptyl
1,1-dimethylheptyl
1,1-dimethylheptyl
1,1-dimethylheptyl
1,1-dimethylheptyl
1,1-dimethylheptyl
–
H
>1000
>1000
>1000
>1000
0.5
2-methyl
1-ethyl
2-ethyl
1-(3,4-dichlorophenyl)
13b
14a
15a
18.6ꢀ4.1
514ꢀ355
5.2ꢀ6.0
1-(2,4-dichlorophenyl)
>40000
ND[b]
>1000
–
SR141716
WIN55,212-2
–
–
7.3ꢀ0.9
–
45.6ꢀ8.6
3.7ꢀ0.2
–
[a] Values obtained from competition curves using [3H]CP55940 as radioligand for hCB1 and hCB2 cannabinoid receptors and are expressed as the mean ꢀ
SEM of at least three experiments. [b] Not determined. [c] Selectivity ratio for CB1 versus CB2.
tration of 40 mm. A complete dose–response curve was gener-
ated for compounds that displaced the radioligand by >50%
in the preliminary screen. Table 1 lists the experimental binding
affinities (Ki values) from the respective displacement curves
for hCB1 and hCB2 receptors.
The first series to be examined were the 2,4-
dihydrochromen[4,3-c]pyrazol-9-oles and 1,4-dihydrochromen-
[4,3-c]pyrazol-9-oles bearing an n-pentyl side chain (com-
pounds 7–10a). Excluding 10a, which binds weakly to the CB1
cannabinoid receptor, the binding data for 7, 8a, 8b, 9a, and
9b (Table 1) clearly show that these compounds bind neither
the CB1 nor CB2 receptor. As reported,[33] the introduction of a
C1’-alkyl substituent to D9-THC, D8-THC, and CBN derivatives
Figure 2. Effect of WIN55,212-2 (WIN), arachidonyl-2-chloroethylamide
leads to an enhancement in affinity for both the CB1 and CB2
receptors. Interestingly, the 1,1-dimethylheptylchromenopyra-
zole derivatives 11, 12b, 13a, 13b, and 15a showed signifi-
cant to high affinity for CB1 (Ki: 4.5–28.5 nm) whereas they did
not bind CB2 at all (Ki: >40000 nm). This is the first observation
of such CB1 receptor selectivity among the cannabinoid ligands
with a classical cannabinoid structure. Thus, chromenopyra-
zoles 11, 12b, 13a, 13b, and 15a show an optimal CB1 selec-
tivity relative to the CB1/CB2 binding data reported for D9-THC,
D8-THC, and CBN derivatives.
(ACEA) and compounds 11, 13a, and 13b in mouse vas deferens. Values
represent the mean ꢀSEM (n=6–8) of modification of the electrically in-
duced contraction of vas deferens tissue by the addition of increasing con-
centrations of vehicle (control), WIN, ACEA, or test compounds. Significant
differences versus control are indicated: *p<0.05, **p<0.01, ***p<0.001
(two-way ANOVA followed by Bonferroni’s post-hoc test).
WIN55,212-2 (WIN); however, their effect was similar to that of
arachidonyl-2-chloroethylamide (ACEA), a CB1-selective agonist
commonly used to characterize cannabinoid effects. Consider-
ing that compound 13a showed the most interesting profile
as potential CB1 agonist, the inhibition of its effect was tested
by adding the cannabinoid antagonist AM251 to the organ
bath 10 min before the addition of the tested compound. As il-
lustrated in Figure 3, the effect of 13a was clearly decreased
by this CB1-selective antagonist. Moreover, the CB2-selective
antagonist AM630 was similarly tested, and it did not block
the effect of compound 13a (data not shown).
With respect to substitution on the pyrazole ring, the Ki
values remained generally similar among the 1,1-dimethylhep-
tyl analogues, except for the N1-3,4-dichlorophenyl moiety:
compound 14a exhibited decreased affinity for both CB1 and
CB2 receptors, with a loss of CB1 selectivity.
Isolated tissue assays
The functional activity of 11, 13a, and 13b was tested on
mouse vas deferens, a tissue where CB1 cannabinoid receptors
are expressed, and thus which is commonly used to study and
characterize cannabinoid effects, as previously described.[34]
Compounds 11, 13a, and 13b inhibited the electrically evoked
contractile response of this tissue. In agreement with its high
affinity for the CB1 receptor, 13a exhibited the highest effec-
tiveness (Figure 2). These three ligands were less effective than
In vivo bioassays
Cannabinoid tetrad
Psychoactive cannabinoids dose-dependently modify sponta-
neous activity, antinociceptive response, rectal temperature,
and catalepsy in mice.[35] Effects of WIN and 13a on the canna-
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Chromenopyrazoles
Figure 5. Antinociceptive effect of compound 13a (1 and 3 mgkgꢁ1), i.p. ad-
ministered, 30 min before HS injection. Bars show the total number of
shakes (mean ꢀSEM); *p<0.05, **p<0.01 versus vehicle (Veh); one-way
ANOVA, nꢂ6.
Figure 3. Effect of AM251 in blocking the activity of compounds 13a. Values
represent the mean ꢀSEM (n=6) inhibition of electrically induced contrac-
tion of mouse vas deferens induced by the addition of increasing concentra-
tions of compound 13a in control tissues or in tissues incubated with
AM251. Significant difference versus control tissues: *p<0.05 (two-way
ANOVA followed by Bonferroni’s post-hoc test).
Molecular modeling
The CB1 cannabinoid receptor selectivity observed for the 1,1-
dimethylheptylchromenopyrazoles offers the opportunity to
explore structural features required for CB1/CB2 selectivity by
molecular modeling. Conformational analysis of N-H-chrome-
nopyrazole 11 and the two N-methylchromenopyrazole re-
gioisomers 13a and 13b was first performed to determine the
global minimum-energy conformation of each, as well as other
minimum-energy conformations. With respect to N-H-chrome-
nopyrazole 11, two tautomers (11 a and 11 b, Figure 6) were
considered. Although conformational analysis of 11 a shows
that it is more stable than 11 b by 1.9 kcalmolꢁ1, both tauto-
mers were taken into consideration for docking studies.
Figure 7 illustrates the global minimum-energy conformers
of tautomers 11 a and 11 b, and regioisomers 13a and 13b.
The global energy minima of these four compounds were then
docked by using a model of the active state (R*) of the canna-
binoid receptors CB1 and CB2.[36,37] These models include the
extracellular and intracellular
binoid tetrad were evaluated. Intraperitoneal (i.p.) administra-
tion of WIN (at 2.5 and 5 mgkgꢁ1) induced antinociceptive
effect in the hot-plate test, hypothermia, catalepsy, and a de-
crease in locomotor activity, whereas compound 13a (at 5 and
10 mgkgꢁ1) did not modify any of these signs of cannabinoid
tetrad activity (Figure 4). This result suggests that 13a lacks
any significant CNS effects.
Orofacial pain model
Hypertonic saline (HS) injection in the masseter of rats pro-
duced a paw-shaking behavior. This nociceptive behavior was
decreased by i.p. administration of compound 13a (at 1 and
3 mgkgꢁ1), suggesting that 13a is active in this pain model
(Figure 5), acting at CB1 receptors located outside the CNS.
loops, the N terminus (truncated
in CB1) and the C terminus, in-
cluding the intracellular helix
portion of each receptor, termed
helix 8. CB1 and CB2 receptor
docking studies were performed
in the same binding site de-
scribed for HU210[38] and for
AM841[39,40] respectively.
Chromenopyrazole–cannabinoid
receptor docking studies
Tautomer 11b–CB1R*
The energy-minimized 11 b–
CB1R* complex is illustrated in
Figure 8. Lys3.28(192) was used
as the primary interaction site
Figure 4. Effect of WIN55,212-2 (WIN) (2.5 and 5 mgkgꢁ1) and compound 13a (5 and 10 mgkgꢁ1) in the mouse
cannabinoid tetrad. Mice were tested for analgesia by A) hot-plate assay, B) rectal temperature, C) catalepsy on a
ring, and D) locomotor activity via rotarod test. **p<0.01, ***p<0.001 versus vehicle (Veh); one-way ANOVA,
nꢂ10.
for CB1 docking studies reported
herein.[41] The phenolic oxygen
atom of 11 b is engaged in a hy-
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455

N. Jagerovic et al.
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molꢁ1), dominated by van der Waals interactions. The energy
difference between the initially docked 11 b conformation and
the final conformation in the energy-minimized complex was
found to be 6.69 kcalmolꢁ1 at the Hartree–Fock (HF) 6-31G*
level. The overall interaction energy for 11 b at CB1 was found
to be ꢁ56.52 kcalmolꢁ1 (see table S1 in the Supporting Infor-
mation).
Figure 6. Structures of the two tautomers 11 b and 11 a.
Tautomer 11a–CB1R*
The docking of tautomer 11 a in the CB1R* receptor model re-
vealed a similar occupation of the binding site, with similar hy-
drogen bonds involving Lys3.28(192) and Ser7.39(383) as
shown in Figure 8. As for the 11 b–CB1R* complex, 11 a has the
greatest pairwise interaction energy with Lys3.28(192)
(ꢁ11.02 kcalmolꢁ1),
and
significant
interactions
with
Cys7.42(386) (ꢁ6.22 kcalmolꢁ1), Asp2.50(163) (ꢁ6.03 kcal
molꢁ1),
Leu7.43(387)
(ꢁ5.20 kcalmolꢁ1),
Asn7.45(389)
(ꢁ5.13 kcalmolꢁ1), and Val3.32(196) (ꢁ4.40 kcalmolꢁ1). Howev-
er, the interaction energy with Ser7.39(383) (ꢁ10.88 kcalmolꢁ1
)
in the 11 a–CB1R* complex was found to be much stronger
than for 11 b. The energy difference between the initially
docked 11 a conformation and the final conformation in the
energy-minimized complex was observed to be 6.51 kcalmolꢁ1
at the HF 6-31G* level. The overall interaction energy for 11 a
at CB1 was found to be ꢁ63.92 kcalmolꢁ1 (see table S1 in the
Supporting Information). Taken together, the energies of inter-
action of 11 a and 11 b at CB1 suggest that 11 a is the preferred
tautomeric form for 11 binding at CB1.
Figure 7. Minimum-energy conformers of tautomers 11 a, 11 b, 13a, and
13b.
Compound 13b–CB1R*
In the energy-minimized 13b–CB1R* complex as illustrated in
Figure 9, 13b forms two hydrogen bonds with Lys3.28(192).
The first involves Lys3.28(192) as hydrogen donor to the phe-
nolic oxygen atom of 13b [H-bond (NꢁO) d=2.75 ꢄ, a(NꢁHꢁ
O)=1528]). The second interaction involves Lys3.28(192) hydro-
gen bonding with the pyrazole N2 atom [H-bond (NꢁN) d=
3.10 ꢄ, a(NꢁHꢁN)=1328]. This pyrazole N2 atom also forms a
hydrogen bond with Ser7.39(383) [H-bond (NꢁO) d=3.15 ꢄ,
a(OꢁHꢁN)=1348]. However, this interaction is weaker than in
the CB1R* complex with 11 and 13a. The ligand 13b has its
Figure 8. Binding site of 11 b (left, in pink) and 11 a (right, in pink) in the
CB1R* model. The amino acid residues interacting with the ligand are shown
in grey. Yellow dashed lines indicate hydrogen bonding interactions.
drogen bond with Lys3.28(192) as reported for the HU210–
CB1R* binding model [H-bond (NꢁO) d=2.78 ꢄ, a(NꢁHꢁO)=
1508]. The N2-pyrazole nitrogen atom is involved in a hydrogen
bond with Ser7.39(383) [H-bond (NꢁO) d=3.15 ꢄ, a(OꢁHꢁ
N)=1428]. Ligand 11 b exhibits the greatest pairwise interac-
tion energy with Lys3.28(192) (ꢁ11.88 kcalmolꢁ1), followed by
Leu7.43(387) (ꢁ5.64 kcalmolꢁ1), Cys7.42(386) (ꢁ5.61 kcal
molꢁ1), and Asn7.45(389) (ꢁ5.43 kcalmolꢁ1). Coulombic energy
dominates the overall pairwise energy of interaction in the
case of Lys3.28(192), whereas van der Waals energy is predomi-
nant for Leu7.43(387), Cys7.42(386), and Asn7.45(389). The in-
teraction with Ser7.39(383) was found to be only ꢁ4.48 kcal
molꢁ1, indicating a weak hydrogen bond with the N2-pyrazole
nitrogen. The tautomer 11 b also has significant interactions
with Asp2.50(163) (ꢁ4.99 kcalmolꢁ1) with van der Waals and
coulombic contributions, and with Val3.32(196) (ꢁ4.97 kcal
Figure 9. Binding site of 13a (left, in pink) and 13b (right, in pink) in the
CB1R* model. The amino acid residues interacting with the ligand are shown
in grey. Yellow dashed lines indicate hydrogen bonding interactions.
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Chromenopyrazoles
greatest pairwise interaction energy with Lys3.28(192)
(ꢁ14.34 kcalmolꢁ1, mainly coulombic energy), followed by
Asn7.45(389) (ꢁ5.19 kcalmolꢁ1), Leu7.43(387) (ꢁ5.13 kcal
molꢁ1), and Cys7.42(386) (ꢁ4.76 kcalmolꢁ1), which are predom-
inantly through van der Waals interactions. The 13b–CB1R*
complex also has significant interactions with Val3.32(196)
(ꢁ4.72 kcalmolꢁ1) and Asp2.50(163) (ꢁ4.70 kcalmolꢁ1). The
energy difference between the initially docked conformation
of 13b and the final conformation in the energy-minimized
complex was found to be 5.13 kcalmolꢁ1 at the HF 6-31G*
level. The overall interaction energy for 13b at CB1 was found
to be ꢁ59.92 kcalmolꢁ1 (see table S2 in the Supporting Infor-
mation).
zole moiety of the structures and the ionic lock as illustrated in
Figure 10. This result suggests that the presence of the pyra-
zole plays a key role in the selectivity of compounds 11 and 13
for the CB1 cannabinoid receptor.
Compound 13a–CB1R*
Figure 10. Binding site of 13b (left, in pink) and 13a (right, in pink) in the
CB2R* model. The D275–K3.28(109) ionic lock is shown in grey. The red cir-
cles indicate steric clash.
The main interactions of the energy-minimized 13a–CB1R*
complex are shown in Figure 9. As observed for 11 a, 11 b, and
13b, Lys3.28(192) forms a hydrogen bond with the phenolic
oxygen atom of 13a [H-bond (NꢁO) d=2.70 ꢄ, a(NꢁHꢁO)=
1668]. The N2 atom of the pyrazole in 13a hydrogen bonds
Discussion and Conclusions
with Ser7.39(383), as a hydrogen bond acceptor [H-bond (Nꢁ Although the first generation of classical cannabinoids showed
O) d=2.80 ꢄ, a(OꢁHꢁN)=1608]. Interestingly, an additional
hydrogen bond between the pyran ring oxygen and
Cys7.42(386) was revealed in the 13a–CB1R* complex [H-bond
(SꢁO) d=2.91 ꢄ, a(SꢁHꢁO)=1728]. The significant increase in
pairwise interaction energy with Cys7.42(386) (ꢁ7.01 kcal
molꢁ1) relative to the other complexes presented herein (11 a,
11 b, and 13b) is a consequence of this additional hydrogen
bond. Ligand 13a exhibits the greatest interaction energy with
Lys3.28(192) (ꢁ13.83 kcalmolꢁ1), followed by the hydrogen
potent activity in vivo, they lacked CB1/CB2 selectivity and most
of them are psychoactive. These CB1-mediated side effects are
due to the fact that CB1 receptors are largely expressed in the
CNS. Thus, the unwanted psychoactive effects of cannabinoid
receptor agonists have limited their development as medi-
cines. Because it is known that CB1 receptors are also located
peripherally,[43] there is a growing interest in targeting cannabi-
noid receptors located outside the brain. For this reason, it is
important to develop new non-psychoactive cannabinoids that
do not cross the blood–brain barrier, but act on peripherally
located cannabinoid receptors. The chromenopyrazoles pre-
sented herein were designed in analogy to CBN, which is a
CB1/CB2 cannabinoid ligand. The binding data show that the
1,1-dimethylheptyl side chain on this scaffold is necessary for
high affinity. The ligand binding studies resulted in Ki values of
4.5–28.5 nm for the 1,1-dimethylheptyl analogues 11, 12b,
13a, 13b, and 15a at hCB1 receptors. Notably, these 1,1-dime-
thylheptyl analogues do not show affinity for hCB2 receptors
(Ki: >40000 nm). Unlike the major members of the classical
cannabinoid family that lack full selectivity for CB1 or CB2, the
chromenopyrazole structure has a determinant influence on
CB1 selectivity. These results suggest that such selectivity can
be explained by the presence of a pyrazole ring in the struc-
ture. However, the substituent on the pyrazole may play a
major role in the binding to both receptors. Thus, we observed
that the 1-(3,4-dichlorophenyl) substituent (compound 14a)
significantly disrupts CB1 receptor selectivity with a loss of af-
finity for CB1 and a moderate affinity for the CB2 receptor.
As assessed by modeling studies, the 1,1-dimethylheptyl-
chromenopyrazoles 11 a, 11 b, 13a, and 13b revealed a similar
occupation of the HU210/CP55940 binding site in the CB1 re-
ceptor model. The phenolic hydroxy group of 11 a, 11 b, 13a,
and 13b is crucial for the interaction with CB1R*, due to a hy-
drogen bond with Lys3.28(192). This residue has been shown
to be critical for the binding of both classical and endocanna-
bond interaction with Ser7.39(383) (ꢁ7.47 kcalmolꢁ1
) and
Phe2.57(170) (ꢁ5.30 kcalmolꢁ1). The Phe2.57(170) interaction
has significant van der Waals and coulombic contributions and
seems to have arisen from the interaction of the phenolic ring
hydrogen atoms with the aromatic ring of the phenylalanine.
The 13a–CB1R* complex also has significant interactions with
Leu7.43(387) (ꢁ7.43 kcalmolꢁ1), Asp2.50(163) (ꢁ6.22 kcal
molꢁ1), Asn7.45(389) (ꢁ4.97 kcalmolꢁ1), and Val3.32(196)
(ꢁ4.94 kcalmolꢁ1). The energy difference between the initially
docked 13a conformation and the final conformation in the
energy-minimized complex was found to be 7.39 kcalmolꢁ1 at
the HF 6-31G* level. The overall interaction energy for 13a at
CB1 was found to be ꢁ69.16 kcalmolꢁ1 (see table S2 in the
Supporting Information). Taken together with the results for
13b above, these docking studies indicate that the interaction
of 13a at CB1 is more energetically favorable than those of its
positional isomer.
Compounds 11a–, 11b–, 13a–, and 13b–CB2R*
Compounds 11 a, 11 b, 13b, and 13a were also docked in the
previously reported advanced CB2R* model[37,40,42] at the
AM841 binding site.[40] The CB2 receptor model contains a salt
bridge between D275 (Asp275) in the EC3 loop and K3.28
[Lys3.28(109)]. Docking studies of the chromenopyrazoles 11 a,
11 b, 13a, and 13b revealed a steric clash between the pyra-
ChemMedChem 2012, 7, 452 – 463
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457

N. Jagerovic et al.
MED
binoids.[41] Furthermore, interaction with Ser7.39(383) was
found to be the main one for the pyrazole moiety, particularly
for the 13a–CB1R* complex. The Ser7.39(383) residue has been
reported to be crucial for the binding of the CB1 agonist
CP55940.[38]
analysis was performed on a Waters 2695 HPLC system equipped
with a Photodiode Array 2996 coupled to a Micromass ZQ 2000
mass spectrometer (ESIMS), using a Waters X-bridge C18 column
(3.5 mm, 2.1ꢅ100 mm) and 30 min gradient A: CH3CN/0.08%
1
formic acid, B: H2O/0.05% formic acid, visualizing at l=254 nm. H
and 13C NMR spectra were recorded on a Bruker 300 (300 and
75 MHz) at 258C. Samples were prepared as solutions in deuterat-
ed solvent and referenced to internal nondeuterated solvent peak.
Chemical shifts (d) are expressed in ppm downfield of tetramethyl-
silane. Elemental analyses were determined with a LECO CHNS-932
instrument. Melting points were determined on an MP70 Reichert
Jung Thermovar apparatus and are uncorrected. The purity of final
compounds was determined by HPLC–MS and elemental analyses
performed with the respective aforementioned instrumentation.
While K3.28 in CB1 has been reported to be critical for the
binding of non-classical, classical, and endocannabinoids,[41]
mutation of the equivalent residue in CB2, K3.28(109) has no
effect on the binding of any cannabinoid ligand.[44] An impor-
tant sequence divergence in the EC3 loop of the CB2 model
(TTLSDQVKK) versus the CB1 model (GKMNKLIKT) has been re-
ported.[40] The CB2R* model suggests a salt bridge formed be-
tween Asp275 and Lys3.28(109) that makes K3.28 unavailable
for ligand interaction. In our CB2R* docking, this salt bridge
causes a steric clash with the pyrazole moiety of 11 a, 11 b,
13a, or 13b due to the rigidity and planarity of the heterocy-
cle. Therefore, these results suggest that the pyrazole moiety
in these compounds is responsible for the CB1 selectivity over
CB2.
5-(1,1-Dimethylheptyl)-1,3-dihydroxybenzene[30] (2): BBr3 (1m in
CH2Cl2, 19 mL, 19 mmol) was added to a solution of 5-(1,1-dime-
thylheptyl)-1,3-dimethoxybenzene (0.45 g, 1.9 mmol) in dry CH2Cl2
at ꢁ168C under N2 in the dark. The reaction mixture was allowed
to warm to room temperature and was stirred for 20 h. CH3OH was
then added carefully at 08C until the mixture reached pH 7. The
solvent was removed in vacuo, and the crude was purified by chro-
matography on silica gel (EtOAc) to give the title compound as a
white solid (0.33 g, 73%); mp: 88–918C (988C);[30] 1H NMR (CDCl3):
d=6.35–6.45 (m, 2H, 4-H), 6.16 (m, 1H, 2-H), 4.65 (bs, 2H, OH),
1.45–1.50 (m, 2H, 2’-H), 1.22 (s, 6H, CH3), 1.21–1.10 (m, 6H, 3’-H, 4’-
H, 5’-H), 1.03 (bs, 2H, 6’-H), 0.84 ppm (t, J=6.5 Hz, 3H, CH3);
13C NMR (CDCl3): d=163.2 (3-C), 154.0 (5-C), 111.2 (4-C), 101.3 (2-C),
45.0 (2’-C), 35.3, 31.1, and 23.9 (3’-C, 4’-C, 5’-C), 29.8 (8’-C), 25.1 (6’-
C), 14.8 ppm (7’-C); HPLC–MS: [A, 20!80%], tR =14.0 min, (90%);
MS (ES+, m/z) 237 (100%) [M+H]+; Anal. calcd for C15H24O2: C
76.23, H 10.24, found: C 76.10, H 10.18.
Compounds 13a and 13b are positional isomers of each
other, differing in the placement of an ethyl substituent (N1,
13a; N2, 13b). Interestingly, the interaction energies of 13a
and 13b with CB1 calculated herein follow the same trend as
their CB1 affinities (13a: Eint =ꢁ69.16 kcalmolꢁ1, Ki =4.5 nm
versus 13b: Eint =ꢁ59.92 kcalmolꢁ1, Ki =18.6 nm).
The chromenopyrazoles 11, 13a, and 13b were tested in
functional in vitro assays. Among them, 13a acted as the most
effective cannabinoid agonist. Its effect was significantly and
almost completely inhibited by the CB1 antagonist AM251 but
not by the CB2 antagonist AM630. Compound 13a was then
selected for carrying out behavioral tests in vivo. It did not
induce modifications in any of the tested parameters on the
mouse cannabinoid tetrad, thus discounting CNS-mediated ef-
fects. This lack of agonistic activity in the CNS suggests that
these compounds do not readily cross the blood–brain barrier.
To study other possibilities of antinociception, compound
13a was tested in another pain model. We chose a model of
orofacial pain in rat (nocive stimulation of the masseter by in-
jection of hypertonic saline); in this model it is known that
other drugs such as opioids can induce antinociception via pe-
ripheral mechanisms.[45,46] In this test, 13a was able to decrease
the nociceptive response. Remarkably, a different result was
obtained from the hot-plate test; from these data it could be
suggested that the antinociception induced by compound
13a in the orofacial test may be mediated through peripheral
mechanisms.
5-Hydroxy-2,2-dimethyl-7-pentylchroman-4-one[29] (3): Olivetol
(0.48 g, 2.7 mmol) and 3,3-dimethylacrylic acid (0.27 g, 2.7 mmol)
were added to a mixture of P2O5 (0.23 g, 1.6 mmol) and methane-
sulfonic acid (4.6 mL, 72 mmol) under N2 at room temperature. The
mixture was then irradiated by microwave at 708C in a sealed reac-
tor for 10 min. The mixture was poured onto water/ice and then
extracted with CH2Cl2. The organic layer was dried over MgSO4.
After removal of the solvent, the crude was purified by chromatog-
raphy on silica gel (EtOAc) to give the title compound as an
orange oil (0.34 g, 48%); 1H NMR (CDCl3): d=6.32 (d, J=2.4 Hz,
1H, 8-H), 6.26 (d, J=2.4 Hz, 1H, 6-H), 2.98 (t, J=7.1 Hz, 2H, 1’-H),
2.66 (s, 2H, 3-H), 1.48–1.53 (m, 2H, 2’-H), 1.44–1.38 (m, 2H, 3’-H),
1.41 (s, 6H, OC(CH3)2), 1.30–1.40 (m, 2H, 4’-H), 0.87 ppm (t, J=
6.9 Hz, 3H, 5’-H); 13C NMR (CDCl3): d=191.2 (4-C), 161.9 (5-C), 159.7
(8a-C), 147.6 7-C), 110. 2 (8-C), 109.9 (6-C), 100.1 (4-C), 76.4 (2-C),
48.3 (3-C), 33.3 (1’-C), 30.1 (3’-C), 28.1 (2’-C), 24.8 (OC(CH3)2), 20.7
(4’-C), 12.2 ppm (5’-C); HPLC–MS: [A, 20!80%], tR =17.6 min,
(90%); MS (ES+, m/z) 263 (100%) [M+H]+; Anal. calcd for
C16H22O3: C 73.25, H 8.45, found: C 72.98, H 8.71.
7-(1,1-Dimethylheptyl)-5-hydroxy-2,2-dimethylchroman-4-one
(4): Prepared from 2 (0.37 g, 1.6 mmol), P2O5 (0.18 g, 1.3 mmol),
methanesulfonic acid (2.67 mL, 54 mmol), and 3,3-dimethylacrylic
acid (1.99 g, 19.9 mmol) by following the procedure described for
3. Pale-yellow oil (0.34 g, 40%); 1H NMR (CDCl3): d=6.45 (d, J=
1.6 Hz, 1H, 8-H), 6.37 (d, J=1.6 Hz, 1H, 6-H), 2.71 (s, 2H, 3-H), 1.52–
1.59 (m, 2H, 2’-H), 1.46 (s, 6H, OC(CH3)2), 1.22 (s, 6H, C(CH3)2), 1.19
(bs, 6H, 3’-H, 4’-H, 5’-H), 1.05 (bs, 2H, 6’-H), 0.87 ppm (m, 3H, 7’-H);
13C NMR (CDCl3): d=197.9 (4-C), 163.0 (5-C), 161.7 (8a-C), 156.0 (7-
C), 107.0 (8-C), 106.2 (6-C), 105.7 (4-C), 79.1 (2-C), 48.5 (3-C), 41.0
(2’-C), 39.2 (1’-C), 32.1, 27.1 and 23.0 (3’-C, 4’-C and 5’-C), 30.3
(C(CH3)2), 28.8 (OC(CH3)2), 25.0 (6’-C), 14.5 ppm (7’-C); HPLC–MS: [A,
Experimental Section
Chemistry
Commercially available starting materials and reagents were used
as supplied. Reactions conducted under anhydrous conditions
were performed under N2 atmosphere in solvents dried over CaCl2
(THF) or Na/benzophenone (THF). Microwave-mediated syntheses
were performed using an 800 W Ethos Synth microwave (Milestone
Inc.) and a CEM Biotage microwave. Column chromatography was
performed using silica gel 60 (230–400 mesh). Analytical HPLC–MS
458
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ChemMedChem 2012, 7, 452 – 463

Chromenopyrazoles
80!100%], tR =4.9 min, (97%); MS (ES+, m/z) 318 (97%) [M+H]+;
added to a solution of 5 (10 mg, 0.03 mmol) in EtOH. The reaction
mixture was stirred at room temperature for 16 h. The solvent was
evaporated under reduced pressure. The crude oil was purified by
column chromatography on silica gel (hexanes/EtOAc 2:1) to iso-
late the two isomers 8a and 8b. Compound 8a was obtained as
an orange oil (4 mg, 39%); 1H NMR (CDCl3): d=7.32 (s, 1H, 3-H),
6.50 (d, J=1.3 Hz, 1H, 6-H), 6.27 (d, J=1.3 Hz, 1H, 8-H), 4.11 (s, 3H,
N-CH3), 2.50 (t, J=7.7 Hz, 2H, 1’-H), 1.55–1.44 (m, 8H, 2’-H and
OC(CH3)2), 1.31–1.25 (m, 4H, 3’-H and 4’-H), 0.89 (m, 3H, 5’-H);
13C NMR (CDCl3): d=154.5 (9-C), 149.9 (5a-C), 145.4 (7-C), 132.0 (9b-
H), 130.9 (3-C)), 123.1 (3a-C), 111.4(8-C), 109.5 (6-C), 76.4 (4-C), 41.1
(N-CH3), 38.7 (1’-C), 35.7 (3’-C), 31.4 (2’-C), 27.3 (OC(CH3)2), 22.5 (4’-
C), 14.0 ppm (5’-C); HPLC–MS: [A, 20!80%], tR =15.1 min, (95%);
MS (ES+, m/z) 301 (100%) [M+H]+; Anal. calcd for C19H26N2O2: C
72.58, H 8.33, found: C 72.81, H 8.26. Compound 8b was obtained
Anal. calcd for C20H30O3: C 75.43, H 9.50, found: C 75.52, H 9.64.
5-Hydroxy-3-(hydroxymethylen)-2,2-dimethyl-7-pentylchroman-
4-one[29] (5): Dry 95% NaH (0.14 g, 6 mmol) was added to a solu-
tion of dihydrochroman-4-one 3 (0.17 g, 0.7 mmol) in dry THF
under N2. The reaction mixture was irradiated by microwave at
468C in a sealed reactor for 20 min. Ethyl formate (0.96 mL,
12 mmol) was then added, and the mixture was irradiated for an
additional 20 min at 468C. The solvent was then evaporated under
reduced pressure. H2O was added to the residue. The aqueous so-
lution was extracted with Et2O and then was neutralized with 1m
HCl and extracted with CH2Cl2. The combined organic layers were
dried (MgSO4) and evaporated under reduced pressure. Column
chromatography on silica gel (hexanes/EtOAc 1:1) afforded the title
1
1
compound as a pale-yellow oil (0.12 g, 60%); H NMR (CDCl3): d=
as a pale-yellow oil (6 mg, 58%); H NMR (CDCl3): d=8.31 (s, 1H,
13.48 (d, J=11.1 Hz, 1H, CHOH), 11.36 (s, 1H, 5-OH), 7.34 (d, J=
11.1 Hz, 1H, 10-H), 6.32 (d, J=1.3 Hz, 1H, 8-H), 6.21 (d, J=1.3 Hz,
1H, 6-H), 2.49 (t, J=7.6 Hz, 2H, 1’-H), 1.58–1.65 (m, 2H, 2’-H), 1.56
(bs, 6H, 9-H), 1.30–1.19 (m, 4H, 3’-H and 4’-H), 0.88 ppm (t, J=
7.2 Hz, 3H, 5’-H); 13C NMR (CDCl3): d=189.9 (4-C), 162.3 (5-C), 161.9
(CHOH), 159.3 (8a-C), 155.9 (7-C), 114.7 (3-C), 109.8 (8-C), 108.7 (6-
C), 105.5 (4-C), 78.7 (2-C), 37.1 (1’-C)), 31.8 (3’-C), 30.4 (2’-C), 28.6 (9-
C), 22.9 (4’-C), 14.4 ppm (5’-C); MS (ES+, m/z) 291 (100%) [M+H]+;
HPLC–MS: [A, 80!100%], tR =1.5 min, (99%); MS (ES+, m/z) 318
(97%) [M+H]+; Anal. calcd for C17H22O4: C 70.32, H 7.64, found: C
70.47, H 7.31.
OH), 7.09 (s, 1H, 3-H), 6.42 (d, J=1.1 Hz, 1H, 6-H), 6.34 (d, J=
1.1 Hz, 1H, 8-H), 3.90 (s, 3H, N-CH3), 2.50 (t, J=7.6 Hz, 2H, 1’-H),
1.59 (bs, 6H, OC(CH3)2), 1.50–1.60 (m, 2H, 2’-H), 1.30–1.18 (m, 4H,
3’-H and 4’-H), 0.95–0.84 (m, 3H, 5’-H); 13C NMR (CDCl3): d=153.6
(9-C), 153.1 (5a-C), 145.3 (7-C), 142.6 (9b-C), 124.0 (3a-C), 120.3 (8-
C), 108.7 (6-C), 108.5 (9a-C), 101.5(9a-C), 76.4(4-C), 38.8 (N-CH3),
36.2 (1’-C), 31.5 and 22.5 (3’-C and 4’-C), 30.7 (2’-C), 29.6 (OC(CH3)2),
14.0 (5’-C); HPLC–MS: [A, 20!80%], tR =19.9 min, (96%); MS (ES+,
m/z) 301 (100%) [M+H]+; Anal. calcd for C19H26N2O2: C 72.58, H
8.33, found: C 72.36, H 8.49.
1-Ethyl-1,4-dihydro-4,4-dimethyl-7-pentylchromeno[4,3-c]pyra-
7-(1,1-Dimethylheptyl)-5-hydroxy-3-(hydroxymethylen)-2,2-di-
methylchroman-4-one (6): Prepared from 4 (0.17 g, 0.5 mmol),
NaH (0.16 g, 6.7 mmol), and ethyl formate (1.44 mL, 2.7 mmol) by
following the procedure described for 5. Column chromatography
on silica gel (hexanes/EtOAc 2:1). Pale-yellow oil (0.16 g, 76%);
1H NMR (CDCl3): d=13.48 (d, J=11.6 Hz, 1H, CHOH), 11.28 (s, 1H,
5-OH), 7.34 (d, J=11.6 Hz, 1H, CHOH), 6.46 (d, J=1.6 Hz, 1H, 8-H),
6.35 (d, J=1.6 Hz, 1H, 6-H), 1.58 (bs, 6H, OC(CH3)2), 1.22 (bs, 6H,
C(CH3)2), 1.12–1.28 (m, 6H, 3’-H, 4’-H and 5’-H), 1.03 (m, 2H, 6’-H),
0.83 ppm (t, J=6.7 Hz, 3H, 7’-H); 13C NMR (CDCl3): d=189.8 (4-C),
163.1 (5-C), 162.0 (CHOH), 161.9 (8a-C), 159.1 (7-C), 114.8 (3-C),
107.8 (6-C), 106.6 (8-C), 105.2 (4-C), 78.7 (2-C), 44.4 (2’-C), 39.2 (1’-
C), 32.1, 30.3 and 25.0 (3’-C, 4’-H and 5’-H), 28.8 (C(CH3)2), 28.6
(OC(CH3)2), 23.0 (6’-C), 14.5 ppm (7’-C); HPLC–MS: [A, 20!80%],
tR =22.7 min, (100%); MS (ES+, m/z) 347 (100%) [M+H]+; Anal.
calcd for C21H30O4: C 72.80, H 8.73, found: C 73.07, H 8.64.
zol-9-ol
(9a)
and
2-ethyl-2,4-dihydro-4,4-dimethyl-7-
pentylchromeno[4,3-c]pyrazol-9-ol (9b): A solution of 5 (40 mg,
0.15 mmol) and ethylhydrazine oxalate (20 mg, 0.15 mmol) in EtOH
(3 mL) was irradiated under microwave for 10 min. The solvent was
evaporated, and the crude residue was subjected to chromatogra-
phy on silica gel (hexanes/EtOAc 1:1) to isolate the two isomers 9a
and 9b. Compound 9a was obtained as a pale-yellow oil (6.0 mg,
1
12%); H NMR (CDCl3): d=9.35 (bs, 1H, OH), 7.38 (s, 1H, 3-H), 6.46
(s, 1H, 6-H), 6.45 (s, 1H, 8-H), 4.48 (q, J=7.1 Hz, 2H, NCH2CH3), 2.50
(t, J=7.6 Hz, 2H, 1’-H), 1.66–1.52 (m, 8H, 2’-H and OC(CH3)2), 1.43
(t, J=7.1 Hz, 3H, NCH2CH3), 1.28–1.19 (m, 4H, 3’-H and 4’-H), 0.88
(t, J=6.7 Hz, 3H, 5’-H); 13C NMR (CDCl3): d=155.5 (9-C), 151.8 (5a-
C), 145.6 (7-C), 132.9 (9b-C), 131.7 (3-C), 122.9 (3a-C), 110.4 (6-C),
109.7 (8-C), 103.1 (9a-C), 76.04(4-C), 48.0 (NCH2CH3), 35.7 (1’-C), 31.4
and 22.5 (3’-C and 4’-C), 30.4 (2’-C), 27.2 (OC(CH3)2), 15.9 (NCH2CH3),
14.0 ppm (5’-C); HPLC–MS: [A, 60!100%], tR =1.6 min, (100%);
MS (ES+, m/z) 315 (100%) [M+H]+; Anal. calcd for C23H34N2O2: C
74.55, H 9.25, found: C 74.63, H 9.19. Compound 9b was obtained
as a yellow solid (0.04 g, 61%); mp: 145–1498C; 1H NMR (CDCl3):
d=8.38 (s, 1H, OH), 7.12 (s, 1H, 3-H), 6.42 (d, J=1.3 Hz, 1H, 6-H),
6.30 (d, J=1.3 Hz, 1H, 8-H), 4.17 (q, J=7.1 Hz, 2H, NCH2CH3), 2.50
(t, J=7.1 Hz, 2H, 1’-H), 1.58–1.53 (m, 8H, 2’-H and OC(CH3)2), 1.48
(t, J=7.1 Hz, 3H, NCH2CH3), 1.30–1.19 (m, 4H, 3’-H and 4’-H), 0.95–
0.81 ppm (m, 3, 5’-H); 13C NMR (CDCl3): d=153.5 (9-C), 153.0 (5a-C),
145.2 (7-C)), 142.4 (9b-C), 122.3 (3-C), 119.8 (3a-C), 108.7 (8-C), 108.4
(6-C), 101.6 (9a-C), 76.7 (4-C), 47.0 (NCH2CH3), 36.2 (1’-C), 31.4 and
22.5 (3’-C and 4’-C), 30.7 (2’-C), 29.6 (OC(CH3)2), 15.4 (NCH2CH3),
14.0 ppm (5’-C); HPLC–MS: [A, 60!100%], tR =2.3 min, (100%);
MS (ES+, m/z) 315 (100%) [M+H]+; Anal. calcd for C23H34N2O2: C
74.55, H 9.25, found: C 74.23, H 9.41.
1,4-Dihydro-4,4-dimethyl-7-pentylchromen[4,3-c]pyrazol-9-ol (7):
A solution of 5 (40 mg, 0.13 mmol) and anhydrous hydrazine
(10 mg, 0.26 mmol) in EtOH (3 mL) was irradiated under microwave
for 10 min. The solvent was evaporated, and the crude residue was
subjected to chromatography on silica gel (hexanes/EtOAc 2:1) to
1
obtain 7 as a white solid (20 mg, 59%); mp: 133–1378C; H NMR
(CDCl3): d=8.52 (bs, 1H, OH), 7.31 (s, 1H, 3-H), 6.44 (d, J=1.5 Hz,
1H, 6-H), 6.37 (d, J=1.5 Hz, 1H, 8-H), 2.53 (t, J=7.1 Hz, 2H, 1’-H),
1.90–1.76 (m, 8H, OC(CH3)2 and 2’-H), 1.41–1.31 (m, 4H, 3’-H and
4’-H), 0.86 ppm (t, J=6.9 Hz, 3H, 5’-H); 13C NMR (CDCl3): d=156.1
(9-C), 153.9 (5a-C), 146.3 (7-C), 142.7 (9b-C), 123.4 (3-C), 120.2 (3a-
C), 109.4 (6-C), 108.6 (8-C), 101.7 (9a-C), 77.0 (4-C), 36.6 (1’-C), 31.9
(3’-C), 30.9 (2’-C), 29.9 (OC(CH3)2), 22.9 (4’-C), 14.4 ppm (5’-C);
HPLC–MS: [A, 10!100%], tR =5.9 min, (98%); MS (ES+, m/z) 287
(97%) [M+H]+; Anal. calcd for C17H22N2O2: C 71.30, H 7.74, found:
C 71.01, H 7.47.
1-(3,4-Dichlorophenyl)-1,4-dihydro-4,4-dimethyl-7-
pentylchromeno[4,3-c]pyrazol-9-ol (10b): Prepared from
5
1,4-Dihydro-1,4,4-trimethyl-7-pentylchromeno[4,3-c]pyrazol-9-
ol[29] (8a) and 2,4-dihydro-2,4,4-trimethyl-7-pentylchromeno[4,3-
c]pyrazol-9-ol[29] (8b): Methylhydrazine (7.00 mL, 0.13 mmol) was
(14 mg, 0.05 mmol) and 3,4-dichlorophenylhydrazine hydrochloride
(10 mg, 0.05 mmol) by following the procedure described for 7.
Column chromatography on silica gel (hexanes/EtOAc 2:1) afforded
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10b as an orange solid (7 mg, 31%); mp: 120–1248C; 1H NMR
(CDCl3): d=7.57 (d, J=2.4 Hz, 1H, 2-Hphenyl), 7.49 (s, 1H, 3-H), 7.44
(d, J=8.7 Hz, 1H, 5-Hphenyl), 7.24 (dd, J=2.4 Hz, J=8.7 Hz, 1H, 6-
Hphenyl), 6.52 (d, J=1.2 Hz,1H, 6-H), 6.10 (d, J=1.2 Hz, 1H, 8-H),
2.53–2.45 (m, 2H, 1’-H), 1.60–1.51 (m, 8H, 2’-H and OC(CH3)2), 1.30–
1.22 (m, 4H, 3’-H and 4’-H), 0.93–0.88 ppm (m, 3H, 5’-H); 13C NMR
(CDCl3): d=154.5 (9-C), 150.0 (5a-C), 146.3 (7-C), 142.0 (1-Cphenyl),
137.9 (3-C), 132.9 (3-Cphenyl), 132.2 (9b-C), 131.0 (4-Cphenyl), 129.9 (5-
Cphenyl), 126.0 (2-Cphenyl), 124.9 (3a-C), 123.5 (6-Cphenyl), 111.2 (8-C),
109.7 (6-C), 102.3 (9a-C), 77.2 (4-C), 36.7 (1’-C), 31.5 and 22.5 (3’-C
and 4’-C), 30.3 (2’-C), 27.2 (OC(CH3)2), 14.0 ppm (5’-C); HPLC–MS: [A,
60!100%], tR =2.3 min, (100%); MS (ES+, m/z) 431 (100%) [M+
H]+.
16.3 (2’-C), 14.4 ppm (7’-C); HPLC–MS: [A, 80!100%], tR =
3.08 min, (99%); MS (ES+, m/z) 371 (100%) [M+H]+; Anal. calcd
for C23H34N2O2: C 74.55, H 9.25, found: C 74.63, H 9.19. Compound
13b was obtained as a white solid (23 mg, 61%); mp: 160–1648C;
1H NMR (CDCl3): d=8.32 (s, 1H, OH), 7.13 (s, 1H, 3-H), 6.57 (d, J=
1.5 Hz, 8-H), 6.48 (s, 1H, J=1.5 Hz, 6-H), 4.16 (q, 2H, J=7.2 Hz,
NCH2CH3), 1.60 (s, 6H, OC(CH3)2), 1.56 (bs, 2H, 2’-H), 1.50 (t, 2H, J=
7.2 Hz, NCH2CH3), 1.24 (s, 6H, C(CH3)2), 1.17 (bs, 6H, 3’-H, 4’-H and
5’-H), 1.01–0.96 (m, 2H, 6’-H), 0.86–79 ppm (m, 3H, 7’-H); 13C NMR
(CDCl3): d=153.6 (9-C), 153.2 (5a-C), 152.9 (7-C), 142.7 (9b-C), 122.8
(3-C), 120.3 (3a-C), 106.9 (6-C), 106.7 (8-C), 101.7 (9a-C), 76.4
(OC(CH3)2), 47.4 (NCH2CH3), 44.9 82(2’-C), 32.2, 30.4 and 30.1 (3’-C,
4’-C and 5’-C), 29.3 (C(CH3)2), 25.0 (OC(CH3)2), 23.1 (6’-C), 15.9
(NCH2CH3), 14.5 ppm (7’-C); HPLC–MS: [A, 80!100%], tR =5.6 min,
(98%); MS (ES+, m/z) 371 (100%) [M+H]+; Anal. calcd for
C23H34N2O2: C 74.55, H 9.25, found: C 74.23, H 9.41.
1,4-Dihydro-4,4-dimethyl-7-(1,1-dimethylheptyl)chromeno[4,3-
c]pyrazol-9-ol (11): Prepared from 6 (16 mg, 0.05 mmol) and anhy-
drous hydrazine (0.01 mL, 0.32 mmol) by following the procedure
described for 7. Column chromatography on silica gel (hexanes/
EtOAc 2:1) afforded 11 as an orange oil (7 mg, 41%); 1H NMR
(CDCl3): d=7.32 (bs, 1H, NH), 6.58 (d, J=1.5 Hz, 1H, 6-H), 6.51 (d,
J=1.5 Hz, 8-H), 6.48 (s, 1H, 3-H), 1.63 (bs, 6H, OC(CH3)2), 1.58–1.52
(m, 2H, 2’-H), 1.25 (s, 6H, C(CH3)2), 1.18 (bs, 6H, 3’-H, 4’-H and 5’-H),
1.12–1.05 (m, 2H, 6’-H), 0.83 ppm (t, J=6.7 Hz, 3H, 7’-H); 13C NMR:
d=(CDCl3) 153.7 (9-C), 153.5 (5a-C), 153.4 (7-C), 144.1 (9b-C), 129.1
(3-C), 123.4 (3a-C), 106.8 (8-C), 106.5 (6-C), 101.7 (9a-C), 77.0
(OC(CH3)2), 44.9 (2’-C), 38.4 (C(CH3)2), 32.2, 30.4, and 30.0 (3’-C, 4’-C
and 5’-C), 29.3 (C(CH3)2), 25.0 (OC(CH3)2), 23.1 (6’-C), 14.5 ppm (7’-
C); HPLC–MS: [A, 80!100%], tR =3.1 min, (100%); MS (ES+, m/z)
343 (100%) [M+H]+; Anal. calcd for C21H30N2O2: C 73.65, H 8.83,
found: C 74.01, H, 8.59.
1-(3,4-Dichlorophenyl)-7-(1,1-dimethylheptyl)-1,4-dihydro-4,4-
dimethylchromeno[4,3-c]pyrazol-9-ol (14a): Prepared from
6
(17.0 mg, 0.05 mmol) and 3,4-dichlorophenylhydrazine hydrochlo-
ride (10.0 mg, 0.05 mmol) by following the procedure described for
7. Column chromatography on silica gel (hexanes/EtOAc 2:1) af-
forded 14a as an orange solid (9 mg, 40%); mp: 124–1268C;
1H NMR (CDCl3): d=7.65 (d, J=2.3 Hz, 1H, 2-Hphenyl), 7.50 (s, 1H, 3-
H), 7.43 (d, J=8.5 Hz, 1H, 5-Hphenyl), 7.25 (dd, J=2.3 Hz, J=8.5 Hz,
1H, 6-Hphenyl), 6.66 (d, J=1.6 Hz, 1H, 6-H), 6.24 (d, J=1.6 Hz, 1H, 8-
H), 1.68 (s, 6H, OC(CH3)2), 1.60–1.54 (m, 2H, 2’-H), 1.22 (s, 6H,
C(CH3)2), 1.20–1.09 (m, 6H, 3’-H, 4’-H and 5’-H), 1.08–1.02 (m, 2H,
6’-H), 0.84 ppm (t, J=6.5 Hz, 3H, 7’-H); 13C NMR (CDCl3): d=154.6
(9-C), 154.0 (5a-C), 150.0 (7-C), 142.3 (1-Cphenyl), 135.3 (3-C), 133.2 (3-
Cphenyl), 132.2 (9b-C), 130.4 (4-Cphenyl), 126.5 (5-Cphenyl), 125.4 (2-
Cphenyl), 123.9 (6-Cphenyl), 109.6 (6-C), 108.0 (8-C), 102.4 (9a-C), 76.2
(OC(CH3)2), 44.7 (2’-C), 38.3 (C(CH3)2), 32.1, 30.3 and 25.0 (3’-C, 4’-C
and 5’-C), 28.9 (C(CH3)2), 27.7 (OC(CH3)2, 23.0 (6’-C), 14.5 ppm (7’-C);
HPLC–MS: [A, 80!100%], tR =5.6 min, (98%); MS (ES+, m/z) 487
(100%) [M+H]+.
7-(1,1-Dimethylheptyl)-2,4-dihydro-2,4,4-trimethylchromeno[4,3-
c]pyrazol-9-ol (12b): Prepared from 6 (30 mg, 0.09 mmol) and
methylhydrazine (0.02 mL, 0.34 mmol) by following the procedure
described for 7. Column chromatography on silica gel (hexanes/
EtOAc 2:1) afforded 12b as a yellow solid (13 mg, 42%); mp:
>3008C; 1H NMR (CDCl3): d=8.23 (s, 1H, OH), 7.09 (s, 1H, 3-H),
6.57 (d, J=1.6 Hz, 1H, 6H), 6.48 (d, J=1.6 Hz, 1H, 8-H), 3.90 (s, 3H,
NCH3), 1.63 (s, 6H, OC(CH3)2), 1.65–1.54 (m, 2H, 3’-H), 1.24 (s, 6H,
C(CH3)2), 1.18–1.09 (m, 6H, 3’-H, 4’-H and 5’-H), 1.10–1.04 (m, 2H,
6’-H), 0.83 ppm (t, J=6.7 Hz, 3H, 7’-H); 13C NMR (CDCl3): d=152.2
(9-C), 151.8 (5a-C), 151.6 (7-C), 141.5 (9b-C), 122.9 (3-C), 119.3 (3a-
C), 105.5 (6-C), 105.3 (8-C), 100.2 (9a-C), 76.4 (OC(CH3)2), 43.5
(NCH3), 38.9 (2’-C), 38.0 (1’-C), 31.8, 30.0 and 29.7 (3’-C, 4’-C and 5’-
C), 28.9 (8’-C), 24.6 (OC(CH3)2), 22.6 (6’-C), 14.1 ppm (7’-C); HPLC–
MS: [A, 80!100%], tR =5.9 min, (100%); MS (ES+, m/z) 357 (100%)
[M+H]+.
1-(2,4-Dichlorophenyl)-7-(1,1-dimethylheptyl)-1,4-dihydro-4,4-
dimethylchromeno[4,3-c]pyrazol-9-ol (15a): Prepared from
6
(0.05 mg, 0.16 mmol) and 2,4-dichlorophenylhydrazine hydrochlo-
ride (0.13 g, 0.63 mmol) by following the procedure described for
7. Column chromatography on silica gel (hexanes/EtOAc 2:1) af-
forded 15a as an orange oil (0.06 g, 75%); 1H NMR (CDCl3): d=
7.43 (s, 1H, 3-H), 7.39 (d, J=2.2 Hz, 1H, 3-Hphenyl), 7.27 (dd, J=
2.2 Hz, J=8.7 Hz, 1H, 5-Hphenyl), 7.21 (d, J=8.7 Hz, 1H, 6-Hphenyl),
6.56 (d, J=1.6 Hz, 1H, 6-H), 6.12 (d, J=1.6 Hz, 1H, 8-H), 1.64 (s, 6H,
OC(CH3)2), 1.46–1.41 (m, 2H, 2’-H), 1.16–1.08 (m, 12H, 3’-H, 4’-H, 5’-
H and C(CH3)2), 1.12–0.98 (m, 2H, 6’-H), 0.82 ppm (t, J=6.9 Hz, 3H,
7’-H); 13C NMR (CDCl3): d=154.3 (9-C), 153.6 (5a-C), 151.3 (7-C),
140.3 (1-Cphenyl), 135.3 (2-Cphenyl), 135.0 (4-Cphenyl), 134.4 (3-C), 133.0
(9b-C), 129.8 (3-Cphenyl), 129.6 (5-Cphenyl), 127.3 (6-Cphenyl), 123.4 (3a-
C), 108.8 (6-C), 107.6 (8-C), 102.6 (9a-C), 77.6 (OC(CH3)2), 44.7 (2’-C),
39.1 (C(CH3)2), 32.1, 30.3 and 24.9 (3’-C, 4’-C and 5’-C), 28.9
(C(CH3)2), 28.0 (OC(CH3)2), 23.0 (6’-C), 14.5 ppm (7’-C); HPLC–MS: [A,
20!80%], tR =19.0 min, (99%); MS (ES+, m/z) 487 (100%) [M+
H]+.
7-(1,1-Dimethylheptyl)-1-ethyl-1,4-dihydro-4,4-
dimethylchromeno[4,3-c]pyrazol-9-ol (13a) and 7-(1,1-dimethyl-
heptyl)-2-ethyl-2,4-dihydro-4,4-dimethylchromeno[4,3-c]pyrazol-
9-ol (13b): Prepared from 6 (35 mg, 0.1 mmol) and ethylhydrazine
oxalate (15.00 mg, 0.1 mmol) by following the procedure described
for 9a and 9b. Column chromatography on silica gel (hexanes/
EtOAc 2:1) allowed isolation of the two isomers 13a and 13b.
Compound 13a was obtained as a pale-yellow oil (5.00 mg, 18%);
1H NMR (CDCl3): d=8.77 (bs, 1H, OH), 7.37 (s, 1H, 3-H), 6.58 (d, J=
1.6 Hz, 2H, 6-H), 6.49 (d, J=1.6 Hz, 1H, 8-H), 4.67 (q, J=7.1 Hz, 2H,
NCH2CH3), 1.57 (s, 6H, OC(CH3)2), 1.58–1.47 (m, 2H, NCH2CH3), 1.44
(t, J=7.1 Hz, 3H, 2’-H), 1.23 (s, 6H, C(CH3)2), 1.17 (bs, 6H, 3’-H, 4’-H
and 5’-H), 1.07 (bs, 2H, 6’-H), 0.84–0.78 ppm (m, 3H, 7’-H); 13C NMR
(CDCl3): d=154.6 (9-C), 153.1 (5a-C), 151.67 (7-C), 133.1 (3-C), 132.2
(9b-C), 123.4 (3a-C), 108.8 (8-C), 108.0 (6-C), 103.2 (9a-C), 76.7
(OC(CH3)2), 48.4 (NCH2CH3), 44.8 (2’-C), 38.1 (C(CH3)2), 32.1, 30.4 and
25.01 (3’-C, 4’-C and 5-C), 29.0 (C(CH3)2), 27.7 (OC(CH3)2), 23.0 (6’-C),
Biological studies
Binding evaluation: Membranes from transfected cells expressing
human CB1 or CB2 cannabinoid receptors (RBHCB1M400UA and
RBXCB2M400UA) were supplied by PerkinElmer Life and Analytical
Sciences (Boston, MA, USA). The CB1 receptor membrane protein
concentration was 2.33 or 3.60 pmolmgꢁ1 depending on the
460
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Chromenopyrazoles
batch, and the protein concentration was 8.0 mgmLꢁ1. The CB2 re-
ceptor membrane protein concentration was 5.20 or
To test the involvement of the CB1 and CB2 receptors in the effect
of 13a, this compound was tested in tissues incubated with the re-
spective cannabinoid antagonists AM251 or AM630 (10ꢁ6 m). Con-
centration–response curves for the new compound were construct-
ed in a step-by-step manner as follows: AM251 or AM630 was
added to the organ bath 50 min after the beginning of electrical
stimulation, and 10 min later a dose of 13a was added; its effect
on the electrically induced contractions was monitored 10 min
later. Electrical stimulation was then stopped, the Krebs solution
was replaced, and the cannabinoid receptor antagonist was added
again to determine the effect of the following concentration of the
new compound. This protocol was repeated for every dose of 13a.
Results were expressed as a percentage of inhibition, taking the
mean amplitude of the last five contractions before the first addi-
tion of the agonist as 100%. Each tissue was employed to con-
struct only one concentration–response curve.
6.20 pmolmgꢁ1
, and the protein concentration was 4.0 or
3.6 mgmLꢁ1 depending on the batch. The commercial membranes
were diluted (~1:20) with binding buffer (50 mm TrisCl, 5 mm
MgCl2·H2O, 2.5 mm EDTA, 0.5 mgmLꢁ1 BSA, and pH 7.4 for CB1
binding; 50 mm TrisCl, 5 mm MgCl2·H2O, 2.5 mm EGTA, 1 mgmLꢁ1
BSA, and pH 7.5 for CB2 binding). The final membrane protein con-
centration was 0.4 mgmLꢁ1 incubation volume and 0.2 mgmLꢁ1 in-
cubation volume for CB1 and CB2 receptor assays, respectively. The
radioligand used was [3H]CP55940 (PerkinElmer) at a concentration
of membrane KD ꢅ0.8 nm, and the final volume was 200 mL for CB1
binding and 600 mL for CB2 binding; 96-well plates and tubes nec-
essary for the experiment were previously siliconized with Sigma-
cote (Sigma).
Membranes were resuspended in the corresponding buffer and
were incubated with the radioligand and test compound (10ꢁ4
–
In vivo behavioral studies: Behavioral testing of cannabinoids was
performed to assess psychoactive drug potential, CNS side effects,
as well as medicinal potential. Compound 13a was evaluated in
tests of CNS activity by using the mouse cannabinoid tetrad. The
potential antinociceptive effect was also evaluated in an orofacial
pain model, induced by hypertonic saline (HS).
10ꢁ11 m) for 90 min at 308C. Nonspecific binding was determined
with 10 mm WIN55,212-2, and 100% binding of the radioligand to
the membrane was determined by its incubation with membrane
without test compound. Filtration was performed by a Harvester
filtermate (PerkinElmer) with Filtermat A GF/C filters pretreated
with 0.05% polyethylenimine. After filtering, the filter was washed
with binding buffer (9ꢅ0.2 mL for CB1 and 9ꢅ0.6 mL for CB2),
dried, and a melt-on scintillation sheet (Meltilex A, PerkinElmer)
was melted onto it. Radioactivity was then quantified by liquid
scintillation spectrophotometry (Wallac MicroBeta Trilux, PerkinElm-
er). Competition binding data were analyzed by using GraphPad
Prism software; Ki values are expressed as the mean ꢀSEM of at
least three experiments performed in triplicate for each point.
Animals: ICR male mice (25–30 g) and Wistar male rats (250–300 g),
purchased from Harlan S.A. (Santa Perpetua de Mogoda, Spain),
were used in cannabinoid tetrad and orofacial pain model, respec-
tively. Animals were supplied with food and water ad libitum and
were housed in a temperature-controlled room at 23ꢀ18C under
a standard 12 h light/dark cycle (08:00–20:00); they were housed
in the test room for at least two days before experimentation.
Throughout the experimental procedure, the international ethics
standards for pain-inducing experiments in laboratory animals[50]
and the European Communities Council Directive of 24 November
1986 (86/609 EEC, Nov. 24, 1986) were followed. All animal proce-
dures were reviewed and approved by the Animal Care and Use
Committee of Rey Juan Carlos University.
Isolated tissue assays: Compounds 11, 13a, and 13b were evaluat-
ed with mouse vas deferens preparations. This is a nerve-smooth
muscle preparation that serves as a highly sensitive and quantita-
tive functional in vitro bioassay for cannabinoid receptor agonists.
These ligands induce a concentration-related decrease in the am-
plitude of electrically evoked contractions of the vas deferens by
acting on naturally expressed prejunctional neuronal cannabinoid
receptors to inhibit the release of the contractile neurotransmitters
noradrenalin and ATP provoked by the electrical stimulation.[47,48]
For this study, male ICR mice weighing 25–30 g were used. Mouse
vas deferens were isolated as described by Hughes et al.[49] Tissues
were suspended in a 10 mL organ bath containing 5 mL Krebs so-
lution (NaCl 118 mm, KCl 4.75 mm, CaCl2 2.54 mm, KH2PO4 1.19 mm,
MgSO4 1.2 mm, NaHCO3 25 mm, and glucose 11 mm), which was
continuously purged with 95% O2 and 5% CO2. Tissues were kept
under 0.5 g resting tension at 378C and were electrically stimulat-
ed through two platinum ring electrodes. They were subjected to
alternate periods of stimulation (trains of five rectangular pulses of
70 V, 15 Hz, and 2 ms duration each, were applied every minute)
and rest (10 min). The isometric force was monitored by a MacLab
data recording and analysis system.
Drugs: WIN55,212-2 and compound 13a were dissolved in ethanol
(1 mgmLꢁ1) and subsequently in ethanol and Tween 80 (1:2), after
which the ethanol was evaporated and saline solution added to
reach final concentration.[47] All solutions were made fresh before
each experiment.
Cannabinoid tetrad: The classical cannabinoid tetrad was per-
formed to study CNS side effects; this test evaluates antinocicep-
tion, hypothermia, catalepsy, and locomotor activity in the same
animal 20 min after cannabinoid administration.[51] Separated
groups of mice (nꢂ10) were i.p. treated with vehicle, WIN55,212-2
(2.5 and 5 mgkgꢁ1) and compound 13a (5 and 10 mgkgꢁ1). Tests
were consecutively conducted with an interval of 5 min between
them.
Antinociception: The hot-plate test was carried out using a hot
plate at 558C as nociceptive stimulus. The latency time for licking
of the front paw was taken as an index of nociception. The latency
was measured before treatment (control latency) and after every
treatment (latency after treatment). The cutoff time was 30 s, and
analgesia was quantified with the formula of the maximum possi-
ble effect (MPE), expressed as a percentage:
The effect of the synthetic cannabinoid agonists arachidonyl-2-
chloroethylamide (ACEA), WIN55,212-2, and that of the new com-
pounds 11, 13a, and 13b (10ꢁ7–1.8ꢅ10ꢁ5 m) was tested by con-
structing concentration–response curves for them in a step-by-step
manner. Curves were carried out by the following protocol: ACEA,
WIN55,212-2, or the new compounds were added at a dose to the
organ bath 50 min after the beginning of electrical stimulation,
and their effect on the electrically induced contractions was evalu-
ated 10 min after addition. Electrical stimulation was then stopped,
the Krebs solution was replaced, and the following dose of the
compounds was added. This protocol was repeated for every dose.
%MPE=ðlatency after treatmentÞꢁðcontrol latencyÞ ꢅ100.
ðcutoff timeÞꢁðcontrol latencyÞ
Hypothermia: Core temperatures in mice were measured using a
P6 thermometer and a lubricated rectal probe (Cibertec, Madrid,
Spain) inserted into the rectum at a constant depth of 1 cm. Data
were recorded before and 30 min after treatment.
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N. Jagerovic et al.
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Catalepsy: Catalepsy was measured using a modified “ring test”,
originally described by Pertwee.[51] Mice were placed on a rubber-
coated metal ring (1=6 cm) fixed horizontally at a height of
30 cm. The amount of time in which the mouse is immobile after
placement on the ring is recorded for 5 min, and it is considered
as an index of catalepsy.
cutoff, and a 4.0 ꢄ hydrogen bond cutoff were used in each stage
of the calculation; 7000 steps of conjugate gradient minimization
in 500-step increments (six times) followed by 1000-step incre-
ments (four times) were employed in a distance-dependent dielec-
tric. A 100 kJmolꢁ1 restraint was placed on all f and y angles in
TMH1–7 and helix 8, and a 50 kJmolꢁ1 restraint was placed on the
Lys3.28(192)–phenolic OH hydrogen bond.
Locomotor activity: Motor coordination was assessed with the ro-
tarod test (Cibertec, Madrid, Spain), in which mice were required to
walk against the motion of a rotating drum with a constant speed
of 10 rpm over the course of 5 min. The time (s) taken to fall was
recorded as latency. Animals were trained to the rotarod test
before the pharmacological assay. On the day of the drug test, ro-
tarod latencies were measured immediately before the drug or ve-
hicle was given and 30 min after drug injection. In all experiments
a 300 s cutoff time was used; this time was assigned a value of
100% for locomotor activity.
Energy expense assessments for docked ligands: To calculate the
energy difference between the global minimum-energy conformer
of each compound and its final conformation after energy minimi-
zation of the ligand–receptor complex, rotatable bonds in the
global minimum-energy conformation were driven to their corre-
sponding value in the final docked conformation, and the single-
point energy of the resultant structure was calculated at the HF 6-
31G* level using Jaguar (implemented in Maestro 8.5, Schrçding-
er).
Orofacial pain model: The injection of 100 mL HS (5% NaCl) in the
masseter of lightly anesthetized rats produces ipsilateral hindpaw
shaking behavior that is accepted as an index of muscle nocicep-
tion.[45,46,52] Separated groups of rats (nꢂ10) were i.p. treated with
the vehicle or compound 13a (1–3 mgkgꢁ1) 30 min after HS was
injected in the masseter. Shaking behavior was quantified by
counting the total number of shakes over a 2 min period after the
intramuscular injection of HS. To count the number of shakes, the
experiments were recorded on video and then played back in slow
motion.
Docking in CB2R*: Global minimum-energy conformations of each
ligand were superimposed on HU210 in its complex with the CB2R*
model (unpublished results). Benzopyran atoms of 14 and 16a/b
were selected for superimposition with benzopyran atoms of
HU210. The conformation of the 1,1-dimethylheptyl side chain in
the HU210–CB2R* complex was used to overlay the 1,1-dimethyl-
heptyl side chain position of 11 and 13a/b. After the superimposi-
tion, HU210 was removed. The pyrazole ring of the chromenopyra-
zoles ligands all had steric clashes with the Asp275/Lys3.28(109)
salt bridge. Changes in side chain dihedrals of these two residues
were attempted to relieve the steric overlaps; however, it was im-
possible to relieve this clash without disrupting this ionic lock.
Molecular modeling
Assessment of pairwise interaction energies: After defining the
atoms of each ligand as one group (Group 1) and the atoms corre-
sponding to a residue that lines the binding site in the final
ligand–CB1R* complex as another group (Group 2), Macromodel
(version 8.6, Schrçdinger, LLC, New York, NY, USA) was used to
output the pairwise interaction energy (coulombic and van der
Waals) for a given pair of atoms. The pairs corresponding to
Group 1 (ligand) and Group 2 (residue of interest) were then
summed to yield the interaction energy between the ligand and
that residue.
Amino acid numbering: The numbering scheme for class A GPCRs
suggested by Ballesteros and Weinstein[53] was employed herein. In
this system, the most highly conserved residue in each TMH is as-
signed a locant of 0.50. This number is preceded by the TMH
number and followed in parentheses by the sequence number. All
other residues in a TMH are numbered relative to this residue.
Conformational analysis of 11 and 13a/13b: Global minimum
energy conformations of 11 and 13a/13b were determined with
Spartan ’04 as follows: the structure of each molecule was built
from the fragment library available in the program. Ab initio
energy minimizations of each structure (HF 6-31G*) were then per-
formed. A conformational search was next performed using Spar-
tan ’04 (Monte Carlo method) followed by a minimization of the
energy of each conformer at the semiempirical PM3 level. For this
search, selected bonds were allowed to rotate: CꢁO bond in the
phenolic ring, the first two CꢁC bonds of the dimethylheptyl chain,
and the NꢁC bond in the ethyl substituent of the pyrazole, in the
case of 13a and 13b. Representative conformers according to
their geometry were selected for ab initio energy minimization (HF
6-31G*), the global minimum-energy conformer of each was used
in docking studies.
Acknowledgements
We gratefully acknowledge research support from Spanish Grant
SAF 2009-12422-C02-02, CANNAB-CM (S-SAL-0261-2006 and
S2010-BMD-2308), and RTA (RETICS RD06/001/0014) to P.G. and
N.J. L.H.-F. and P.M. are respective recipients of a Postdoctoral
Contract (ref. JAEDoc-07-00204) and a JAE-Pre Fellowship (re-
f. JAEPre-2010-01119) from C.S.I.C.
Keywords: agonists · cannabinoids · peripheral · protein
models · receptors
Docking with CB1R*: Binding site anchoring interactions within the
receptor for each ligand were based on earlier published docking
studies for HU210.[38] Lys3.28(192) was used as the primary interac-
tion site for the phenolic hydroxy group of each chromenopyra-
zole. Mutation of this residue in CB1 results in the loss of binding
of classical, non-classical, and endocannabinoids, suggesting that
interaction with K3.28 is crucial for binding of this class of
ligand.[41] The energy of the ligand–CB1R* TMH bundle complex
was minimized using the OPLS2005 force field in Macromodel 9.1
(Schrçdinger Inc., Portland, OR, USA). An 8.0 ꢄ extended non-
bonded cutoff (updated every 10 steps), a 20.0 ꢄ electrostatic
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Received: November 30, 2011
Revised: January 18, 2012
Published online on February 2, 2012
ChemMedChem 2012, 7, 452 – 463
ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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